This application claims priority under 35 U.S.C. xc2xa7xc2xa7119 and 365 to Application No. 11-329,400 filed in Japan on Nov. 19, 1999.
1. Field of the Invention
This invention relates to a DNA fragment for stable expression of an exogenous gene in a plant, a method to introduce an exogenous gene for stable expression of an exogenous gene, and a transgenic plant for stable expression of an exogenous gene.
2. Description of Related Arts
Many kinds of transgenic plants, wherein exogenous genes are introduced, have been produced to render various characteristic to the plants. In the production of such transgenic plants, the diversity of gene expression observed among individual transgenic plants makes some problems. It is considered that such diversity is caused by the introduced position of a gene in a chromosome. When an exogenous gene is introduced into active chromatin region, high expression of the exogenous gene would be obtained. On the contrary, when an exogenous gene is introduced into inert chromatin region, sufficient expression of the gene would not be obtained (Galli, Current opinion in plant technology (1998) 1:166-172, Matzke et al., Current opinion in plant technology (1998) 1:142-148). Such effect described above is called xe2x80x9cposition effectxe2x80x9d. Because of the position effect, an exogenous gene, introduced into a plant, exhibits absolute failure of expression, only weak expression or suppression of expression depending on plant growth or surrounding environment. This phenomenon deserves a barrier on commercialization of a transgenic plant and establishment of a method to stabilize expression of an introduced gene have been demanded.
Recently, some transformants, wherein genes of various kinds are introduced, revealed constant expression of the gene independent of its introduced position in the chromosome. The factors concerning such phenomenon are classified into three cases. These are, insulator and MAR (matrix attachment region). Moreover, involvement of such factors on constant expression is suggested. These three factors function as boundaries in a chromosome and blockade effects of near-existing chromatin, though the mechanisms of action of these factors differ with each other.
MAR, described above, is utilized for stable expression of an exogenous gene introduced into a plant by inhibiting the position effect. MAR contains an adenine, thymine (AT) rich sequence and a topoisomerase II recognition sequence. Moreover, MAR is a functional region exhibiting nuclear-matrix binding activity under in virto condition. MAR is considered to exist more than every 10-100 kb in a chromosome, and the chromosome binds to nuclear matrix through these regions to form conformation of the chromosome. MAR is indispensable for compaction of chromosome in a limited space of nucleus. The knowledge indicating that the MAR might be involved in the regulation of gene expression is accumulating in recent researches.
Moreover, it is speculated that, a chromatin loop is formed among plural MAR""s which assures independence of gene expression in the loop. The effect of MAR, when utilized for stabilization of gene expression, was not consistent among experiments and stable expression of introduced gene was not achieved in many cases. This result might be caused by the following phenomenons.
(1) Plenty of MAR""s might operate to increase expression of introduced gene.
(2) When plural copies are introduced, expression of introduced gene might be suppressed by other effects than the position effect, for example, specific methylation of DNA.
Therefore, MAR may not necessarily function as a boundary on the chromosome (Galli, Current opinion in plant technology (1998)1:166-172, Matzke et al., Current opinion in plant technology (1998)1:142-148), resulting in failure of generalization of the technique using MAR.
On the other hand, the insulator described above is also utilized to inhibit the position effect. Here, an enhancer is a DNA sequence that enhances transcriptional activity of a promoter. On the contrary, a silencer is a DNA sequence that attenuates or vanishes the transcriptional activity of a promoter. In detail, two kinds of mechanisms are reported concerning occurrence of silencer function. These are; the DNA sequence of a silencer or conformational structure of a chromosome (chromatin structure) would be involved in inhibition of transcriptional activity of the promoter. An insulator is defined as a functional region (DNA fragment) which blockades such interference effect caused by adjoining genes such as an enhancer or a silencer. In a higher eukaryotic plant, an enhancer and a silencer might cause an effect on transcriptional activity of not only a certain promoter, but also plural promoters. It should be noted that an enhancer or a promoter can cause its transcriptional activity, even to a gene localized at a distance of several bps. Therefore, plural elements might cause effects randomly in a chromosome. This phenomenon might work to render diversity on gene expression. On the other hand, a mechanism to restrict the enhancer function or the silencer function might be necessary for precise regulation of gene expression. At present, it is speculated that an insulator might operate to restrict these functions.
Hitherto, insulators have been identified from various organisms, for example, Drosophila. The examples of insulators identified are, gypsy insulator, scs-scsxe2x80x2 insulator and Fab-7 insulator originated from Drosophila, beta globin insulator originated from chicken, apoB insulator originated from chicken and human. The identification of proteins involved in insulator function have been performed recently, in particular, the analysis on gypsy insulator, scs-scsxe2x80x2 insulator is advanced. Among these researches, it is reported that, beta globin insulator originated from chicken affects to stabilization on gene expression regardless of the origin of the gene. Moreover, the inventors have showed stable expression of an exogenous gene achieved by ligating an insulator isolated from sea urchin arylsulfatase (Japanese patent application No. 11-253174). On the basis of knowledge described above, this invention was performed with the aim to obtain a novel gene which enables stable expression of an exogenous gene introduced.
This inventors isolated some promoters, which enables high extent of expression, from tobacco cultured cell (Nicotiana tabacum. BY-2). Each of the DNA fragment of the promoter thus obtained was ligated to GUS reporter gene, then introduced into tobacco cultured cell. Moreover, GUS activity of each dependent transformed clone was investigated. As the result, the expression of a promoter, derived from tobacco alcohol dehydrogenase (NtADH) gene, showed only few diversity among plural clones.
From knowledge described above, the inventors have searched a nucleotide sequence, which is responsible for stable expression of a gene, in promoter region of NtADH. The region capable of stable expression a gene according to this invention, designated to ADH200, was thus obtained. The detailed characteristic of ADH200 will be described below. ADH200 is expected to be very useful as a novel technique to stabilize expression of an exogenous gene introduced into a plant and contribute to progression of plant bio-industry.
This invention provides a novel DNA fragment useful for stable expression of an exogenous gene introduced into a plant. The DNA fragment according to this invention is consisted of a base sequence derived from promoter region of tobacco alcohol dehydrogenase (NtADH), corresponding to the region from TATA box to 214 bp upstream of TATA box. The inventors have found that said base sequence stabilizes expression of a gene and designated the base sequence as ADH200. In this specification, the wording xe2x80x9cstabilization of expression of a genexe2x80x9d indicates that the ratio of individuals, exhibiting no activity or extremely low activity of an exogenous gene introduced, is significantly decreased. The base sequence of ADH200 of this invention is specified by SEQ ID No. 1 in a sequence list. Moreover, a DNA fragment consisting of a base sequence with high homology to said ADH200, a part of which is deleted or substituted by another sequence, or to which another sequence is added, is in the range of this invention, so far as the DNA fragment is capable of stabilizing expression of an exogenous gene.
The DNA fragment of this invention includes a DNA fragment consisting of a base sequence that hybridizes with the base sequence referred to as SEQ ID No. 1 in the sequence list under stringent condition.
Moreover, the ADH200 DNA fragment of this invention includes a DNA fragment having a base sequence at least 70% of sequence homology with the base sequence referred to as SEQ ID No. 1 in the sequence list, as far as retaining biochemical characteristic as ADH200 DNA fragment. In preferred form, the base sequence of this invention have more than 80% of sequence homology with the base sequence referred to as SEQ ID No. 1 in the sequence list. In more preferred form, the base sequence of this invention have more than 90% of sequence homology with the base sequence referred to as SEQ ID No. 1 in the sequence list.
A vector comprising a fusion gene, the fusion gene consisting of DNA fragment of ADH200, an exogenous gene to be introduced and an exogenous promoter locating between said DNA fragment of ADH200 and said exogenous gene to regulate expression of said exogenous gene, is also in the range of this invention. That is, a vector, containing a fusion gene, the fusion gene consisting of an exogenous promoter located 5xe2x80x2 upstream of an exogenous gene to be introduced and said ADH200 DNA fragment ligated further 5xe2x80x2 upstream of said exogenous promoter, is also in the range of this invention. Moreover, a vector, containing a fusion gene, the fusion gene consisting of an exogenous promoter located 3xe2x80x2 downstream of an exogenous gene to be introduced and said ADH200 DNA fragment ligated further 3xe2x80x2 downstream of said exogenous promoter, is also in the range of this invention. In this specification, an exogenous promoter means that the origin of the promoter is different from host plant, to which an exogenous gene to be introduced. As described in the following embodiment, the ADH200 DNA fragment stabilizes expression of an exogenous gene by stabilizing expression of an exogenous promoter. Therefore, ADH200 gene can stabilize expression of an exogenous gene introduced, even if ADH200 is located outside of the promoter, which is the most prominent feature of this invention.
As shown in the embodiment, the expression of an exogenous gene can be stabilized by introducing said vector, comprising said ADH200 DNA fragment, an exogenous gene and an exogenous promoter, into a plant. A method to introduce an exogenous gene comprising such process is also in the range of this invention. Moreover, a transgenic plant with an exogenous gene introduced by the method described above to achieve stable expression of the exogenous gene, is also in the range of this invention. As described in the following embodiment, expression of an exogenous gene is stabilized using ADH200 DNA fragment of this invention. That is, the number of individuals, with an exogenous gene introduced failed to express its activity or individuals with the extent of expression of an exogenous gene was very low, decreased significantly.
By using the method of this invention, stable expression of various exogenous gene can be achieved. Theoretically, any gene can be adopted as an exogenous gene to be introduced in a host plant. The examples of exogenous genes preferred to be introduced in a host plant are as described below. These are disease or insect injury resistance genes such as peroxidase gene or chitinase gene, genes for ectoine biosynthesis such as L-2,4-diaminobutyric acid acetyltransgerase, L-2,4-diaminobutyric acid transaminase and ectoine synthetase, genes for betaine biosynthesis such as choline oxidase and second metabolite producing gene such as fatty acid biosynthesis. Moreover, an exogenous gene can be introduced into various host plants according to the method of this invention, theoretically, any plant can be adopted as a host plant to be introduced an exogenous gene. The examples of plants preferred to be adopted as a host plant are as described below. These are benetificial cultivated plants such as tobacco, Arabidopsis or petunia, crops such as rice, maize, potato, sweet potato, soybean, strawberry or eggplant and trees such as blue gum or white poplar.
The above description and following example are intended to only illustrate this invention, not to be intended to limit the range of this invention.